Project Summary The goal of this project is to develop a novel drug, R-4329, for prevention of Clostridium (C.) difficile infection (CDI). R-4329 is a probiotic engineered to recombinantly express and orally deliver a unique microbial protein known as secreted antigen A (SagA) which our team showed can protect against multiple enteric infections, including C. difficile, by enhancing the integrity/functionality of the gut membrane barrier1, 2. C. difficile is a ubiquitous anaerobic Gram-positive bacterium that can sporulate and become highly resistant to environment stresses and frontline antibiotics such as clindamycin7. Pathogenic strains of C. difficile secret toxins (A and B) that damage intestinal epithelial cells, resulting in inflammatory colitis, severe diarrhea, abdominal pain, flu-like symptoms, and possible death7. C. difficile infection (CDI) often occurs following antibiotic-treatment when the endogenous microflora of individuals is altered or severely reduced. Since 2000, the rate of CDI has increased from below 150,000 to over 250,000 cases and has been become one of the most significant hospital-acquired infections with an economic burden of over $1 billion to treat in the U.S.7. Once C. difficile establishes residence in the colon, it is difficult to eradicate with last resort antibiotics (vancomycin) and consequently results in lifelong relapses of CDI and inflammatory colitis. An effective approach to treating CDI has been fecal microbiota transplant (FMT) therapy but this approach is heterogeneous and a poorly defined therapeutic, for which its safety is still a concern requiring special FDA approval8. New and well-defined therapeutics are desperately needed to prevent and treat recurrent CDI. Oral probiotics offer an exciting alternative to manage CDI and strains of Lactobacillus (acidophilus, casei, reuteri, plantarum) have been explored to inhibit enteric pathogen infection and mitigate antibiotic-associated diarrhea9. Unfortunately, mechanism of action has been difficult to understand with modest beneficial effects in the context of their clinical utility10. However, these recent studies suggest that improved probiotics with directed and targeted anti-infective functionality may be useful at controlling CDI. Indeed, our recent results published in the journal Science demonstrate that engineering or ?reprogramming? of probiotics to recombinantly express SagA yields a more protective functionality against enteric infections, including CDI2. Our goal is to develop a novel probiotic strain to deliver recombinant SagA (referred to as R-4329) as an orally administered drug that functions naturally to induce protective regulatory signals. This Phase II SBIR application is intended to build upon success and advance R-4329 towards clinical testing. The specific aims are: 1) optimize R-4329 upstream process development and create a GMP master cell bank, 2) manufacture R-4329 and develop GLP analytical product release/stability assays, 3) complete R-4329 dose optimization and biomarker-based PD studies in mice, 4) perform R-4329 GLP toxicology studies in mice to support our IND filing.